Mouse embryonic stem (ES) cells are continuous cell lines derived directly from the fetal founder tissue of the preimplantation embryo. They can be expanded in culture while retaining the functional attributes of pluripotent embryo cells. In particular, they can participate fully in fetal development when reintroduced into the embryo. The capacity for multilineage differentiation is reproduced in culture whose embryonic stem cells produce a wide range of well-defined cell types. Such pluripotent stem cells can constitute a renewable source of more differentiated cells that can be employed to replace diseased or damaged tissue by cellular transplantation.
Mouse ES cells were established from 129 strain mouse blastocysts in 1981, Evans M J, Kaufman M. Nature 292: 154–156 (1981); Martin G. R. Proc. Natl. Acad. Sci. USA 78: 7634–7638 (1981). They can be cultured and manipulated in vitro and then introduced back into the embryonic environment to differentiate into somatic and germ cell lineages even after transfection. The protocols for ES cell derivation include embryos at the expanded blastocyst stage being plated, either intact or following immunosurgical isolation of the inner cell mass (ICM) onto a feeder layer. Roberson E. J. Trends Genet. 2:9–13 (1986).
ES cells injected into host embryos give rise to mosaic mice known as chimeras. Male ES cells are injected into unsexed blastocysts. If the host embryo is female and the male ES cells make germ cells, the chimera will often be a fertile male. If the proportion of ES cell descendents in the coat of the animal is high, the probability that ES cells are represented in gametes is also high, since ES cells mix thoroughly with host cells early in embryogenesis. ES cells give rise to brown coat color because they are Aw/Aw (dominant White-bellied Agouti), and the host cells give rise to black coat color because they are a/a (recessive non-agouti). The ES cells are from the 129 strain of mice; the host embryos are from the C57BL6 strain of mice. If the chimeras are bred to a/a non-agouti mice (for example C57BL6 or Black Swiss), then any brown offspring (Aw/a) must have arisen from ES cell-derived gametes, and 50% of the brown offspring are expected to carry the knockout allele.
The differences in chimerism are due to different amounts of contribution of the ES cells to the Blastocysts. The better the ES cells do in the blastocyst the more cells of the embryo are derived from the ES cells. The most important lineage is the germ line, because that is the only way to pass on the genetic information to the next generation (germline transmission).
An important characteristic of ES cells is that even after extended propagation and manipulation in vitro, ES cells remain capable of re-entering embryogenesis, colonize the germ cell lineage into a chimera and generate functional gametes. Therefore, genetically modified mice can be derived from cultured ES cells. For example, the following types of genetically modified mice may be produced from genetically modified ES cells: 1) knockout mice in which a gene is dysfunctioned, 2) knockin mice in which a gene is introduced in a specific site of a gene of interest, 3) site-specific transgenic mice which is similar to the knockin but certain copies of the gene are merely introduced into a certain site. These are mosaics, with the exogenous gene in only a proportion of their cells and crossing these mosaic animals generates fully genetically modified offspring. 4) non-site-specific transgenic mice which is similar to the conventional transgenic mice but no pronuclear microinjection is employed.
Maintaining ES cell pluripotency and germline transmission are two most important factors to be addressed for generating genetically modified mice. For example, germline-competent ES cell lines require early passage of ES cells and addition of LIF in the culture medium, and conditions that contribute not only ES pluripotency but also germline transmission. The combination of ES cells and embryo is also a very important factor affecting germline transmission.
A large number of ES cell lines have been generated since 1981 and some of the commonly used ES cell lines include the CCE, HM-1, E14, AB1 and R1 lines, all these ES cell lines derived from 129-sub strains or 129 hybrids. Smith A. G, Annu Rev Cell Dev. Biol 17: 435–462, 2001. The preference for gene targeting in pure inbred lines has now dictated a demand for ES cell lines from various inbred strains, such as C57, Balb/c and DBA etc.
There is a special need for genetically modified C57 mice as experimental tools because in some medical fields, the C57 mice are more sensitive than other mice such as the 129 strain, e.g. in electrophysiology and neuroscience. For example, the C57 mice are more sensitive to tastants than 129 mice, and therefore, the C57 mice are preferred when studying taste transduction.
However, C57 ES cells are difficult to get germline transmission because they are much easier to differentiate than the 129 ES cells.
The chimerism of the chimeras is commonly evaluated by coat color, eg., if 129 ES cells are injected into black C57 blastocysts, the offspring, if chimeric, will be a black background (from C57 blastocysts), with agouti chimerism. The above example is the currently commonly used method for making genetically modified mice. If pure C57 background is preferred, normally researchers need to backcross the F1 offspring (50% 129; 50% C57) (which are commonly from the mating of chimeras with C57 mice, see FIG. 3) with C57 mice, this will take about 2 years after 8–10 generations of mating with C57 mice. This makes it very time-consuming to generate genetically modified C57 mice. The generation of gene-targeted mice via C57 ES cells has become an invaluable research tool. Balb/c mice and 129 mice were used to be host blastocysts for the C57 ES cells, the combination C57 ES cells with Balb/c blastocysts results in significantly higher frequencies of chimeras than the combination C57 ES cells with 129 blastocysts. Ledermann, B. and K. Burki, Exp. Cell Research 197:254–258 (1991). The main disadvantage of using Balb/c as donors is that it is difficult to get good quality and quantity of blastocysts from Balb/c mice because of their delayed embryonic development.
Some researchers have used albino C57 blastocysts (C57BL/6J-Tyrc-2J) and introduced C57 ES cells into them with good result. C57BL/6J-Tyrc-2J strain has a white coat color but still C57 black background, it has a G to T base change at nucleotide 291 of the TyrC-2J allele and resulting in an amino acid change from arginine to leucine at residue 77 which lies in the highly conserved DDRE sequence. Le Fur et al, Genomics 37:245–248 (1996). The disadvantage is that albino C57BL/6J-Tyrc-2J mice are expensive and not available in large quantities for experimentation because only one vendor, the Jackson Laboratory, in Maine, provides this strain in limited quantity.
The present invention overcomes the disadvantages outlined above and provides methods for producing germline-competent C57 ES cell lines, and genetically modified (knockout, knockin or transgenic) C57 mice.